METHODS: This paper introduces a biological inspired fuzzy adaptive window median filter (FAWMF) which computes the fuzzy membership strength of nucleotides in each slide of window and filters nucleotides based on median filtering with a combination of s-shaped and z-shaped filters. Since coding regions cause 3-base periodicity by an unbalanced nucleotides' distribution producing a relatively high bias for nucleotides' usage, such fundamental characteristic of nucleotides has been exploited in FAWMF to suppress the signal noise.
RESULTS: Along with adaptive response of FAWMF, a strong correlation between median nucleotides and the Π shaped filter was observed which produced enhanced discrimination between coding and non-coding regions contrary to fixed length conventional window filters. The proposed FAWMF attains a significant enhancement in coding regions identification i.e. 40% to 125% as compared to other conventional window filters tested over more than 250 benchmarked and randomly taken DNA datasets of different organisms.
CONCLUSION: This study proves that conventional fixed length window filters applied to DNA signals do not achieve significant results since the nucleotides carry genetic code context. The proposed FAWMF algorithm is adaptive and outperforms significantly to process DNA signal contents. The algorithm applied to variety of DNA datasets produced noteworthy discrimination between coding and non-coding regions contrary to fixed window length conventional filters.
FINDINGS: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples.
CONCLUSIONS: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.
RESULTS: Using two independent gene-prediction pipelines, Fgenesh++ and Seqping, 26,059 oil palm genes with transcriptome and RefSeq support were identified from the oil palm genome. These coding regions of the genome have a characteristic broad distribution of GC3 (fraction of cytosine and guanine in the third position of a codon) with over half the GC3-rich genes (GC3 ≥ 0.75286) being intronless. In comparison, only one-seventh of the oil palm genes identified are intronless. Using comparative genomics analysis, characterization of conserved domains and active sites, and expression analysis, 42 key genes involved in FA biosynthesis in oil palm were identified. For three of them, namely EgFABF, EgFABH and EgFAD3, segmental duplication events were detected. Our analysis also identified 210 candidate resistance genes in six classes, grouped by their protein domain structures.
CONCLUSIONS: We present an accurate and comprehensive annotation of the oil palm genome, focusing on analysis of important categories of genes (GC3-rich and intronless), as well as those associated with important functions, such as FA biosynthesis and disease resistance. The study demonstrated the advantages of having an integrated approach to gene prediction and developed a computational framework for combining multiple genome annotations. These results, available in the oil palm annotation database ( http://palmxplore.mpob.gov.my ), will provide important resources for studies on the genomes of oil palm and related crops.
REVIEWERS: This article was reviewed by Alexander Kel, Igor Rogozin, and Vladimir A. Kuznetsov.